Muscles must be able to relax as well as contract. Contraction and relaxation are regulated by molecular switches on the thick (myosin-containing) and thin (actin-containing) filaments, which together make up the contractile machinery. Defects in regulation can lead to muscle disease. Our long term goal is to understand the structural basis of muscle regulation; our focus in this application is the relaxed state. During the current grant period we achieved two key breakthroughs in defining the molecular structure and regulatory mechanism of myosin filaments, resolving basic questions dating back more than 40 years. We also gained new insights into troponin-tropomyosin regulation of actin filaments. Our insights raise key new mechanistic questions, which we propose to investigate in this renewal. To understand how muscles relax, the structures of the actin and myosin filaments in the relaxed state must be determined. To achieve this, state-of-the-art electron microscopic and 3D image reconstruction techniques will be applied to native filaments and their isolated component molecules. Using these approaches: (1) The molecular interactions underlying the relaxed state of striated and smooth muscle myosin filaments will be defined in detail. (2) The specific amino acid residues and protein domains essential to these relaxed-state interactions will be determined by analyzing the effects of targeted mutations on the structure of expressed single myosin molecules. (3) The structural basis of thin filament relaxation will be defined by analyzing the native, relaxed-state organization of the regulatory components troponin and tropomyosin, and the thin filament template protein, nebulin. In each study, reconstructions will be fitted to atomic resolution crystal structures of thick and thin filament subunits, defining molecular contacts at near-atomic resolution. Our studies of myosin molecules and filaments will provide new insights into general mechanisms of thick filament regulation, and especially into the nature of the relaxed state in vertebrate striated muscle. Further advances in our studies of troponin-tropomyosin regulated thin filaments will provide new information on native thin filament structure, the 3D organization of troponin and tropomyosin, and the structural contribution of nebulin to the relaxed state. Preliminary data demonstrate the feasibility of our aims. The new insights arising from these proposals will provide a deeper understanding of the structural basis of muscle relaxation. Defining molecular mechanisms in healthy muscle is essential to understanding structural defects that underlie muscle disorders. PUBLIC HEALTH RELEVANCE: Muscles must be able to relax as well as contract. Defects in relaxation lead to diseases such as hypertension and cardiomyopathy. Insights into the molecular basis of relaxation that emerge from our work will lay the foundation for understanding the cause of such diseases, and also how muscles are activated.